Pyroelectric enzyme detector

ABSTRACT

A pyroelectric sensor is provided for detecting enzymes or enzymatic substrates in a liquid or gas stream. The sensor contains a laminate formed of a pyroelectric material sandwiched between upper and lower conducting means to act as electrodes. An electronic circuit means is connected to the conducting means to measure thermally induced electric current and an enzyme or enzymatic substrate is immobilized on a surface of one of the conducting means. The laminate is supported by a support means and a plate means rests on an upper surface of the supported laminate. The plate means has a longitudinal flow channel communicating with the upper surface for flow of a gas of liquid, and a recess for accommodating the electronic circuit means. Inlet and outlet means pass through the plate means and communicate with the flow channel.

BACKGROUND OF THE INVENTION

This invention relates in general to the detection of enzymes orenzymatic substrates directly by measuring the thermal signals producedby their reaction on the surface of a pyroelectric material.

The detection and measurement of enzymes or enzyme substrates inbiological fluids is a problem of considerable importance in thedetection of disease conditions or the elucidation of metabolic pathwaysin animal and vegetable substances. Workers in this field have beenchallenged by the need to develop an analytical method or instrumentthat is sensitive and reliable as well as adaptable to the physicalconstraints that may be imposed upon an analytical technique to measureenzyme systems in biological fluids. Many approaches have beeninvestigated, including the detection of temperature changes in theenvironment of an enzyme and substrate resulting from the heat ofreaction between that pair. Others have tried to detect temperaturechanges with thermistors and other temperature sensing devices but themethods have lacked sensitivity. Pyroelectric gas sensors have beendisclosed by Taylor, U.S. Pat. No. 3,861,879, and Zemel, U.S. Pat. No.4,551,425. The Taylor invention is limited to a device for the detectionof a particular gas and is specifically directed to the detection ofcarbon monoxide. The Zemel invention incorporates a heater in apyroelectric substrate to desorb adsorbed substances and corrollatestheir detection from heat changes associated with change of state. Zemelalso describes and claims application of his pyroelectric sensor for thedetection of an enzyme or coenzyme by reaction with a correspondingenzyme or coenzyme bound to the sensor. The pyroelectric sensor of Zemelis found to possess less than desirable sensitivity. Pyroelectricsensors, such as those described by Zemel, experience shortcomings whenthe enzyme or coenzyme are insufficiently bound to the sensor or wherethe bonding process significantly deactivates the enzyme.

Workers in the field of enzyme or enzyme substrate detection have alsobeen challenged by the problem of how to immobilize said enzyme orsubstrate on a detector without chemically deactivating either.Similarly, investigators have struggled with the problem of detectingenzyme substrate reactions using thermal sensors where the heat of thereaction between the enzyme and substrate is weak.

Nothing in the prior art satisfactorily discloses an operablepyroelectric sensor for enzyme detection or solves the problemsdiscussed herein.

OBJECTS OF THE INVENTION

In response to the foregoing problems and disadvantages in theapplication of pyroelectric materials to the detection of enzymes andtheir substrates it is an object of the present invention to provide apyroelectric sensor for the detection of a corresponding substrate in agas or liquid stream, having an enzyme deposited and immobilized withoutsignificant deactivation on the pyroelectric surface.

A further object of this invention is to provide an improvedpyroelectric sensor for detecting an enzyme in a gas or liquid streamsuch as a biological fluid having deposited and immobilized on thepyroelectric surface the substrate for said enzyme.

Yet another object of this invention is to provide a pyroelectric sensorof improved sensitivity having deposited thereon chemicals reactive tothe products of reaction of enzyme/substrate components.

A further object of this invention is to provide a pyroelectric sensorof improved sensitivity by utilizing materials of preferred thermalconductivity.

Yet another object of the present invention is to provide an improvedmethod to deposit an enzyme on the surface of a pyroelectric sensor insuch a manner that the enzyme is immobilized without significantdeactivation.

SUMMARY OF THE INVENTION

The foregoing objectives are accomplished through the following:

A pyroelectric sensor for detecting an enzyme or enzymatic substrate ina gas or liquid stream comprising:

a. a pyroelectric material;

b. first means, affixed to first face of said pyroelectric material, forconducting an electrical response from said pyroelectric material;

c. at least one reactant selected from the group consisting of an enzymeand an enzymatic substrate reactable respectively with a correspondingsubstrate or enzyme in a gas or liquid stream to produce a thermallydetectable endothermic or exothermic reaction, said reactant beingimmobilized in contact with said first conductive means;

d. second means, affixed to second face of said pyroelectric material,for conducting an electrical response from said pyroelectric material;

e. electronic circuit means connected to said first and secondconducting means to measure thermally induced electrical current fromsaid pyroelectric material;

f. support means to contain said pyroelectric material.

To further summarize the invention, on one of the electrodes of apyroelectric sensor one or more of an enzyme or enzymatic substrate isdeposited and chemically immobilized thereon. The laminate ofpyroelectric material sandwiched between said first and secondconducting means is installed in a structure so designed as to permit astream of gas or liquid containing the material to be detected to flowover the surface on which the enzyme or substrate is immobilized. Anelectrical connection is made from one electrode to the input of acurrent to voltage converter and thence to a voltage amplifier so as toallow analog scaling of the signal. The second electrode of the laminateis connected to ground using a suitable conductor. In this way thematerial to be detected reacts with the reactant deposited on theelectrode to produce a heat of reaction subsequently detectable ascurrent flow in a pyroelectric material.

In another embodiment of the present invention both a substrate orenzyme and a chemical reactant reactable with the reaction products of asubstrate/enzyme system are deposited on the electrode adjacent the zonewhere that specific substrate or enzyme reaction produces a heat ofreaction insufficient to measure with accuracy. The heat of reactionbetween the enzyme/substrate reaction products and the depositedchemical will enhance the thermal signal of the overall reaction andincrease the electrical signal from the pyroelectric material to a levelmore readily detectable with accuracy.

A further embodiment of this invention utilizes a support structureconstructed from materials characterized by low thermal conductivity andhigh heat capacity. Such materials provide a minimum heat sink for thethermal signal produced by the enzyme substrate reaction and therebydoes not deleteriously affect the detection of that signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the pyroelectric sensor of theinvention;

FIG. 2 is a side view of the support structure with the pyroelectriclaminate installed thereon;

FIG. 3 is a top view of the interior of the support structure;

FIG. 4 is a perspective view of the backing of the support structure;and

FIG. 5 is a diagram illustrating the voltage temperature response for anenzyme/substrate analysis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings and with specific reference to FIG. 1, alayer of pyroelectric material 1 such as polyvinylidene flouride (PVD)is coated on its top and bottom sides with aluminum 2. It should beunderstood that any pyroelectric material can be employed in thisinvention and any electrically and thermally conductive metal may beused in place of aluminum to act as electrodes.

In order to electrolytically protect the aluminum electrodes a smallquantity of gold film 3 is sputtered onto and over the surface of thealuminum. The electrodes are connected to a suitable converter source 4to convert the current to voltage and to amplify the voltage formeasurement. A reactant such as enzyme 5 is deposited and immobilized onto one surface of the sensor for subsequent reaction with the materialto be detected, in this case an enzymatic substrate. The enzymedeposited and immobilized pursuant to the present invention can be anyenzyme known to react with enzymatic substrates to produce a thermalchange. Illustrative of such an enzyme are catalase, urease, carbonicanhydrase, arginase, allantolase and the like.

Where it is desired to detect and/or measure the concentration of anenzyme in a gas or liquid stream, an enzymatic substrate specific to theenzyme is deposited and immobilized on the electrode. Thus, thesubstrate or a derivative thereof must be chemically immobilizable.Suitable enzymatic substrates or derivatives thereof include urea forthe detection of urease, peroxides for the detection of catalase, etc.

As aforementioned, where the thermal effect of an enzyme/enzymesubstrate reaction is low, its detection can be enhanced by causing theproducts of reaction between said enzyme and substrate to react with achemical entity or reactant deposited on the same electrode adjacent tothe reactant enzyme or substrate. The chemical reactant can be anychemical capable of reacting with enzyme/enzymatic substrate reactionproducts to produce a thermal change. For example, urease can bedeposited on said electrode for reaction with urea present in a liquidstream. The reaction product, ammonia, subsequently reacts with analdehyde deposited adjacent to the urease, preferably downstream of theenzyme/substrate reaction zone on the electrode. The strongammonia-aldehyde reaction increases the thermal signal from theurease-urea reaction. The reactive aldehyde may be benzaldehyde or apolymeric molecule containing aldehyde functionality such aspolyacrolein.

While there are a variety of methods available to immobilize enzymes andsubstrates on electrode surfaces the preferred approach in the contextof this invention is to employ an aldehyde such as gluteraldehyde whenimmobilizing the enzymes. Gluteraldehyde reacts to crosslink the enzymeand establish immobility without significantly reducing enzyme activity.To achieve this, the immobilization reaction should be conducted between-10 and +10° C. an the reaction quenched after approximately 10 minutes.The quenching can be effected by using an amino acid or other compoundscontaining primary amine groups.

As shown in FIGS. 2 and 3 the resulting pyroelectric laminate ofpyroelectric material sandwiched between said first and secondconducting means 6 is placed in a support structure comprising a flatbacking member 7 (see also FIG. 4) on which the laminate 6 is supportedand an upper plate 8 (see also FIG. 3) which rests on the laminate 6.The upper plate 8 includes a milled flow channel 10 which is disposeddirectly above laminate 6 and a milled recess 11 for accomodating theconnections to the laminate 6. Flow channel 10 communicates withorthogonal inlet passages 12 and 13 formed in the plate 8 and adapted atthe upper ends thereof to receive respective threaded inlet and outlettubing connectors 14 and 15 therein. Connectors 14 and 15 are connectedto an inlet tube 16 and an outlet tube 17, respectively. A liquid or gasstream containing an enzymatic substrate is introduced through the inlettube 16 into the flow channel 10 in the interior surface of plate 8 incontact with the pyroelectric laminate 6 and exits through the outlettube 17.

A further advantage provided by the pyroelectric sensor of the inventionis that it can be cut and formed into a variety of shapes permitting ahigh degree of manufacturing flexibility and ease of application.Accordingly, the ability to vary the electrode material as well as thepyroelectric material offered by the sensor is deemed an importantadvantage of this invention.

The following example is included to illustrate the construction andutilization of one embodiment of this invention.

EXAMPLE

The following example is included to illustrate the construction andutilization of one embodiment of this invention.

Aluminum coated polyvinylidene fluoride pyroelectric film is obtainedfrom a commercial source and cut to the desired size using caution tokeep the film clean. Care should also be exercised to avoid oneelectrode folding around to the other electrode. One electrode surfaceis coated by sputtering thereon approximately 100 Angstroms of gold. 5Milligrams of catalase is mixed well with 35 milligrams of bovine serumalbumin and 200 microliters of water and the mixture is chilled toapproximately 8° C. 20 Microliters of glutaraldehyde are then added tothe resulting solution and the solution is spread over the gold-coatedaluminum electrode surface. The crosslinking reaction is allowed toproceed for approximately 10 minutes and the sensor is then rinsed witha glycine solution. The thus prepared pyroelectric sensor is placed inthe support structure shown in FIG. 2 and electrical leads attached asshown and approximately 50 microliters of a 0.093 molar solution ofhydrogen peroxide is allowed to flow over the gold surface. The outputfrom the electrode connections are recorded on a logging device througha current to voltage converter and voltage amplyfier. The results arereported in FIG. 5 and show a significant voltage difference detectedwithin 40,000 milliseconds, indicative of a strong catalase peroxidereaction.

Various modifications of the pyroelectric enzyme sensor described abovewill be apparent to those of ordinary skill in the art and are embracedby the scope of the following claims.

What is claimed is:
 1. A pyroelectric sensor for detecting an enzyme orenzymatic substrate in a gas or liquid stream comprising:(a) apyroelectirc laminate having an upper and lower surface comprised of apyroelectric material sandwiched between upper and lower conductingmeans to act as electrodes for conducting an electrical response fromsaid pyroelectric material; (b) electronic circuit means connected tosaid electrodes to measure thermally induced electric current from saidpyroelectric material; (c) a reactant selected from the group consistingof an enzyme and an enzymatic substrate reactable respectively with acorresponding enzyme or substrate to produce a thermally detectableendothermic or exothermic reaction, said reactant being chemicallyimmobilized on a surface on one of said conducting means; (d) a supportmeans supporting said laminate; (e) a plate means resting on the uppersurface of said supported laminate, said plate means having alongitudinal flow channel for said gas or liquid stream disposed aboveand communicating with the upper surface of said laminated and saidplate and means having a recess communicating with said upper surfacefor accomodating said electronic circuit means; (f) an inlet meanspassing through said plate means communicating with said flow channelnear one end thereof; and (g) an outlet means passing through said platemeans communicating with said flow channel near an end opposite saidinlet means.
 2. A pyroelectric sensor according to claim 1 wherein thereactant is an enzymatic substrate.
 3. A pyroelectric sensor accordingto claim 1 wherein the reactant is an enzyme.
 4. A pyroelectric sensoraccording to claim 1 wherein said support means is a material of highheat capacity and low thermal conductivity.
 5. A pyroelectric sensoraccording to claim 4 wherein said support means is a thermoplastic orthermosetting resinous material.
 6. A pyroelectric sensor according toclaim 5 wherein said support means material is styrofoam.
 7. Apyroelectric sensor according to claim 4 wherein said support means is aglass material.
 8. A pyroelectric sensor according to claim 1 whereinsaid upper and lower conducting means is a metal.
 9. A pyroelectricsensor according to claim 1 including a chemical entity in contact withone of said conducting means reactively responsive to reaction productsof said group of an enzyme or an enzymatic substrate to provide adetactable thermal signal.